Project Summary The long term objective of this work is to uncover guidance mechanisms that are critical to axon targeting during development, plasticity, and regeneration following injury. The discrete nature of taste papillae and the taste buds within their epithelium make it an excellent model for studying axon targeting mechanisms. Taste axons innervate taste buds but not adjacent epithelium, and somatosensory axons that penetrate non-taste epithelium surround the taste bud but do not enter it. It is also likely that both types of axons recognize non-target and specific target cells within their target zones, further limiting exploration. Surprisingly little is known about the molecules and mechanisms that underlie targeting in this system. Diffusible molecules such as Sema3A and neurotrophins have roles in repelling axons from and attracting axons toward epithelial targets, respectively, but are unlikely to fully account for the ability of taste and somatosensory axons to distinguish between adjacent populations of target and non-target epithelial cells. Cell-attached cues could provide the signaling resolution needed for discrimination between adjacent target and non-target cell populations. EphA receptors (EphA's) and ephrinA's are cell-attached cues that can mediate growth promotion, repulsion (non-target recognition), or stabilization (target recognition). The first aim of the proposed work is to determine which ephrinA's and EphA's are expressed in lingual gustatory papillae and in the sensory nerves that innervate them during embryonic development. Multiple approaches will be used to localize these proteins: in situ hybridization, immunohistochemistry, detection using synthetic proteins containing the extracellular domain of their binding partners, and detection of marker proteins substituted for EphA's or ephrinA's in mutant mice. Second, to determine if EphA/ephrinA signaling promotes or repels taste and somatosensory neurites, ganglion explants will be grown on substrates that have been micro-patterned with alternating stripes of ephrinA's or EphA's and control proteins. To determine if EphA's or ephrinA's are necessary for normal targeting, nerve trajectories in the tongues of mutant mice lacking multiple ephrinA's and/or EphA's will be evaluated. Identifying guidance molecules and understanding their roles in targeting axons to papillae and taste buds may uncover guidance mechanisms that apply to other parts of the nervous system in which the fidelity of initial innervation is not as obvious as in the tongue. These mechanisms may also be employed throughout life as peripheral epithelial targets routinely undergo turnover in the absence of injury and are also re-innervated after injury.